Figure 1.
Expression levels and transcriptional activity of ERβ in BG-1. A.
BG-1 cells were infected with Ad5 or Adβ adenoviruses and treated for 24h with control vehicle ethanol (Control) or E2 10−8M (E2). The expression of ERβ and rS9 reference gene was measured by real-time PCR. Results represent the mean ± SD of ERβ expression normalized by rS9 of 3 independent experiments. Measurements of Adβ and Adβ+E2 groups were compared by unpaired Student's t test. ** p<0.001. B. Proteins were extracted from cells infected in the same conditions as in A and treated for 0, 3, 6 or 24h with E2 10−8M (E2). Levels of ERα and ERβ were analyzed by western blot. Actin was used as a loading control. The upper band of ERβ blot labeled with a star corresponds to aspecific staining. C. Transcriptional activity of ERβ. BG-1 cells were infected with Ad5 or Adβ adenoviruses and transfected with ERE2-TK-Luc reporter along with β-galactosidase reporter. The cells were treated with ethanol as vehicle (Control) or E2 (10−8M) for 24h. Results show relative Luc activities (% of values of Ad5-infected cells without E2) ± SD after normalization with β-gal activity (3 independent experiments). Measurements of Ad5+E2 and Adβ+E2 groups were compared by unpaired Student's t test. * p<0.05. D. PEO14 cells were infected with Ad5, Adα, Adβ or the combination of Adα and Adβ adenovirus and transfected with ERE2-TK-LUC reporter along with β-galactosidase reporter. Cells were treated with control vehicle (Control) or E2 (10−8M) for 24h. Results show relative luciferase activities (% of values of Ad5 infected cells without E2) ± SD after normalization with β-gal activity (3 independent experiments). Measurements of Adα, Adβ and Adα+β groups were compared by unpaired Student's t test. *** p<0.001. NS: non significant. E. Proteins from PEO14 cells infected in the same conditions as in D and treated with vehicle ethanol (Control) or E2 10−8M (E2) for 3, 6 or 24h were analyzed by western blot with antibodies against ERα and ERβ. Actin was used as a loading control. The upper band labeled with a star in right panel corresponds to aspecific staining.
Figure 2.
ERβ is a negative regulator of BG- cell growth. A.
The growth of BG-1 cells, expressing or not ERβ, was monitored in vitro using a cell counter. BG-1 cells were plated in 24-well plates and cultured in the presence of vehicle or E2 (10−8M). Proliferation is expressed as % of control cells grown at day 0. Data represent the mean ± SD from triplicates. Measurements of Ad5+E2 and Adβ+E2 groups were compared by unpaired Student's t test. ** p<0.001. B. ERβ inhibits tumor growth in a bioluminescent subcutaneous mouse model. BG-1 cells stably expressing Luc and infected with Ad5 or Adβ adenoviruses were injected subcutaneously in ovariectomized female Nude mice. Luciferase activity was monitored for 25 days. Results are expressed in photons/s (Ph/s) and represent the mean ± SD from 8 animals. Measurements of Ad5+E2 and Adβ+E2 groups were compared by unpaired Student's t test. * p<0.05.
Figure 3.
ERβ disturbs cell cycle of BG-1 cells and regulators. A.
BG-1 cells were collected 24h after infection with Ad5 or Adβ adenoviruses and analyzed for cell cycle distribution. Results represent the mean ± SEM of 3 experiments. Ad5 and Adβ groups were compared by unpaired Student's t test. ** p<0.001. B. BG-1 cells were infected with Ad5 or Adβ viruses. After infection, cells were treated for 3, 6 or 24h with vehicle (ethanol, C) or 10−8M E2. 30 µg of protein extracts were used for Western blot. β-actin was used as a loading control. Unless specified, the ratio of target proteins over β-actin is indicated below the gels. Representative of 2 experiments.
Figure 4.
ERβ inhibits tumor growth in an orthotopic xenograft mouse model. A.
BG-1-Luc cells infected with Ad5 or Adβ adenoviruses were injected in the left ovary of Nude mice. Luc activity was monitored for 35 days as described in Fig. 1C. Results are expressed in photons/s (Ph/s) and represent one representative experiment corresponding to the average ± SD of at least 5 animals per group. Mann-Whitney test was used for comparison. * p<0.05 and ** p<0.01. A representative image of day 35 is shown. B. Representative pictures of whole animals and genital tract of animals euthanized at day 35 are displayed.
Figure 5.
ERβ reduces metastasis and improves survival. A.
Nude mice injected with BG-1-Luc cells in the same conditions as in Fig. 4. were euthanized 28 days after injection. Lung, liver and right contralateral ovary were taken and Luc activity was assayed as mentioned above. Results are expressed as Photons/s/mg of proteins and represent the mean of 5 animals ± SEM. Mann-Whitney test was used for comparison. * p<0.05 and ** p<0.01. B. Kaplan-meier survival curve. 10 mice were orthotopically xenografted with BG-1 cells stably expressing Luc, infected with Ad5 or Adβ adenoviruses, followed-up daily for the development of respiratory distress, limb paralysis and weight loss, and euthanized immediately if noted. P value is the one obtained in log-rank tests.